: Significance: N-linked protein glycosylation represents an important class of biomolecules receiving significant attention for their application in disease diagnosis and therapeutic development. A major hurdle with rapid and facile N-glycan structural identification stems from the lack of well-defined reagents that can be used as bioanalytical reference material. The inability to robustly interpret protein N-glycosylation prevents development of new technologies for uncovering biomarkers and hinders the advancement of recombinant glycoprotein engineering, such as antibodies. Current commercially available reference material either provides a limited number of N-glycans which are not representative of biological composition or lack details regarding exact structural assembly. Our library will comprise the largest commercially available collection of well-defined N- glycans and will represent a significant advancement to current available standards. Strategy: Taking advantage of the strict substrate requirements of glycosyltransferases and hydrolases, an enzymatic approach will be utilized to produce a collection of high purity structures. Specific Aim: This project describes a robust semi- synthetic, enzymatic methodology for producing the 20 most abundant N-glycans found in human serum all having an abundance greater than 1%, and together, comprise greater than 50% of the total serum N-glycome. Each compound will have a purity of >99% and will contain a free-reducing anomeric terminus providing flexiblity for use in a multitude of analytical techniques. Task 1: Extraction of a sialylated glycopeptide from commercially available egg yolk powder and the preparation of an advanced intermediate which is common to all desired targets. Task 2: Synthesis of bi-antennary targets with symmetric and asymmetric architectures. Task 3: Synthesize a collection of tri-antennary, sialylated regio-isomers. Task 4: Development of a second product line where each N-glycan contains one uniformly labeled 13C6 GlcNAc which can be used as an internal reference for quantification. Preliminary Data: A scalable extraction strategy has been devised for accessing gram- quantities of the desired sialylated glycopeptide starting material from egg yolk powder using robust extraction technology. The synthetic methodologies to be employed in tasks 1 ? 4 have been validated removing the need for further optimization. Deliverables: When complete, the described library will represent the most comprehensive collection of structurally defined N-glycan standards and will deliver an urgently needed tool to the scientific community.